1. Field of the Invention
The present invention relates generally to the fields of fluorimetry and optical sensors. More specifically, the present invention relates to a filterless device to measure fluorescence lifetime of a fluorophore or of an optical sensor to detect a chemical parameter.
2. Description of the Related Art
Fluorescence sensing is based on excitation of a sensor and measurement of its emission characteristics. This includes fluorescence intensity and fluorescence lifetime. Because excitation and emission occur at different wavelengths, it is possible to resolve them using optical filters. However, any filtering decreases the intensity of the emission which is usually quite low. Additionally, filters are not able to completely exclude the excitation wavelengths. Furthermore, as filters are the most expensive component in an optical system and as it is difficult to integrate filters with electronics and/or chemical sensors, the cost of the device is significantly increased.
Another possibility to discriminate between excitation and emission is to use their time characteristics. If a probe or optical chemical sensor is excited by a light with variable intensity, this results in fluorescence which has well defined characteristics and lags in time. For example, if the probe is excited by narrow pulses of light to create delta-like function pulses, the emission intensity is a series of decay curves. If the detector is turned on a short time after the excitation source is turned off completely, however, the delay, or gate time, prevents scattered excitation light and the emission of short lifetime fluorophores from being detected (FIG. 1). This is referred to as gated detection.
The delta-function-like pulses are achievable using lasers having very short pulse duration and very high peak power. However, the use of lasers in sensing is impractical because of the high cost and volume of the instrument. Laser diodes could be an alternative, however, they are still significantly more expensive than a typical light emitting diode (LED).
Although an LED is almost an ideal excitation source in having a bandwidth up to 100 MHz, a narrow emission spectrum of ˜40 nm and efficiency of 5 MW optical power at 40 mA DC, a typical LED does not have sufficient optical power to create delta-function-like pulses. An alternative is to use square wave modulation. The increase of the pulse width increases the signal amplitude. If the period of the excitation is long enough, the starting point of the florescence decay is much closer to the theoretical maximum as determined by the probe quantum yield and concentration. Gating during the excitation pulse from a LED, using a wider gate to accommodate the width increase of the pulse, produces an output signal similar to that using gated impulse excitation from a laser (FIG. 2). However, now the decay curves are separated by a significant time interval equal to half of the period of the excitation light where the output of the photodetector equals zero.
In frequency domain or phase-modulation fluorimetry when the excitation light source is sinusoidally modulated in intensity, intensity of the emission follows the same pattern. That is the emission fluorescence is at the same circular frequency as the excitation light. However, as the fluorophore's excited state is of finite duration, fluorescence lifetime creates a time lag which appears as a phase shift of angle φ and a decrease in depth of modulation as compared to the circular frequency of the excitation light. A demodulation factor, m, is defined by:m=(B/A)/(b/a)where “A” is the average value of the emitted fluorescence, “a” is the average value of the excitation light, “B” is the amplitude of the peak emission above its average value, and “b” is the amplitude of the peak excitation above its average value (FIG. 3).
The circular frequency of the excitation light is expressed:ω=2πfwhere f is the excitation frequency in Hertz. The demodulation factor m, which corresponds to the reduction in the depth of modulation compared to that of the excitation, and the phase angle φ can be measured and used to calculate the modulation lifetime:τm=(ω−1)[(1/m2)−1]1/2and the phase lifetime:τp=(ω−1)(tan φ).
The acquired decay curves contain lifetime information for the investigated fluorophore, ambient light and noise. Under strict experimental conditions it is possible to eliminate the ambient light by using a black box and to eliminate the noise through integration. However, in sensors this is hardly possible as the sensing pad is always exposed to some light. Thus phase-modulation fluorimetry methods perform poorly when there is leakage of the excitation or when the lifetimes in a sample significantly differ. The leakage distorts the information rendering it almost useless (FIGS. 4A/4B).
Thus, the inventors have recognized a need in the art for improvement in phase-modulated fluorimetry and in discriminating the lifetime fluorescence of a fluorophore or of an optical sensor of interest in a sample from any background or other fluorophore. The prior art is deficient in as much as the lack of a device that can successfully measure fluorescence lifetime of an optical sensor without using filters. Specifically, the prior art is deficient in the lack of a filterless device for sensing fluorescence lifetimes of a fluorophore using a combination of gated fluorescence detection and phase-modulation fluorimetry. The present invention fulfills these long-standing needs and desires in the art.